Treatment with melatonin after onset of experimental uveitis attenuates ocular inflammation.

BACKGROUND AND PURPOSE: Uveitis is a prevalent intraocular inflammatory disease and one of the most damaging ocular conditions. Pretreatment with melatonin prevented ocular inflammation induced by an intravitreal injection of bacterial LPS in the Syrian hamster. Here, we have assessed the anti-inflammatory effects of melatonin administered after the onset of ocular inflammation. EXPERIMENTAL APPROACH: The eyes of male Syrian hamsters were intravitreally injected with vehicle or LPS. Melatonin was injected i.p. every 24 h, starting 12 or 24 h after the LPS injection. A clinical evaluation (with a score index based on clinical symptoms), the number of infiltrating cells, protein concentration and PGE2 and PGF2α levels in the aqueous humour, as well as retinal NOS activity, lipid peroxidation and TNF-α levels were assessed. Retinal function was assessed by scotopic electroretinography, and light microscopy and immunohistochemistry were used to evaluate the state of the retinal structure. KEY RESULTS: Both treatment regimens with melatonin decreased clinical symptoms, reduced the leakage of cells and proteins, and decreased PG levels in aqueous humour from eyes injected with LPS. In addition, melatonin treatment blocked the decrease in scotopic electroretinogram a- and b-wave amplitude, protected the retinal structure and reduced the increase in NOS activity, lipid peroxidation and TNF-α levels, induced by LPS. CONCLUSIONS AND IMPLICATIONS: These results indicate that treatment with melatonin, starting after the onset of uveitis, attenuated ocular inflammation induced by LPS in the Syrian hamster and support the use of melatonin as a therapeutic resource for uveitis treatment.

AGE formation blockade with aminoguanidine does not ameliorate chronic allograft nephropathy.

AIMS: Advanced glycation end products (AGEs) are produced by glycoxidation and lipid peroxidation. AGEs induce oxidative stress and inflammation, and accumulate in tubular cells after kidney transplantation. We hypothesize that the AGE formation blocker aminoguanidine (AG) reduces AGE formation and improves renal transplant function. MAIN METHODS: Fisher 344 kidneys were orthotopically transplanted into Lewis recipients. Recipients were treated with AG (100 mg/kg/day), candesartan (CAND; 5mg/kg/day), or vehicle (VEH) for 24 weeks. The major non-cross linking AGE N(ε)-carboxymethyllysine (CML) was measured post-transplantation with gas chromatography-tandem mass spectrometry or immunohistochemistry. As a marker of systemic lipid peroxidation 8-isoprostane was measured by ELISA. We determined intra-arterial blood pressure, heart weight/body weight ratio, size of cardiomyocytes and cardiac hypertrophy as assessed by echocardiography. For biochemical evaluation of cardiac and renal fibrosis we measured hydroxyproline content. KEY FINDINGS: AG significantly reduced serum CML and 8-isoprostane, but did not reduce signs of chronic allograft nephropathy (CAN) or blood pressure. AG did not alter tubular AGE accumulation. AG reduced heart weight/body weight ratio (AG: 2.7 ± 0.1g/kg; CAND: 2.2 ± 0.1, VEH: 3.0 ± 0.4 g/kg), size of cardiomyocytes (P < 0.05) and showed a tendency to reduce cardiac hypertrophy (wall volume average radial AG 7.072 ± 0.83 cm(3) vs. CAND 6.841 ± 0.66 cm(3) vs. VEH 7.839 ± 0.74 cm(3)). SIGNIFICANCE: Despite effective reduction of serum CML and 8-isoprostane, AG did not ameliorate CAN or reduce renal AGE accumulation. On the other hand AG reduced cardiac size suggesting a supportive cardio-protective action which is blood pressure independent.

Leukotriene B4 and 8-isoprostane in exhaled breath condensate of children with episodic and persistent asthma.

BACKGROUND: Leukotrienes and isoprostanes are biomarkers of airway inflammation and oxidative stress that can be detected in exhaled breath condensate (EBC). The aim of this study was to evaluate leukotriene B4 (LTB4) and 8-isoprostane levels in EBC of healthy and asthmatic children with episodic and moderate persistent asthma. METHODS: EBC was collected from 62 children aged 6 to 14 years: 22 healthy children, 30 patients with episodic asthma, and 10 patients with moderate persistent asthma, without preventive treatment at the time of enrolment. RESULTS: LTB concentrations were higher in children with asthma than in healthy controls (50.7 pg/mL vs. 13.68 pg/mL, P < .011). The same was true for children with moderate persistent asthma compared to children with episodic asthma (146.9 pg/mL vs. 18.85 pg/mL, P < .0001), children with moderate persistent asthma compared to healthy controls (146.9 pg/mL vs. 13.68 pg/mL, P < .0001), and children with episodic asthma compared to healthy controls (P, nonsignificant). EBC concentrations of 8-isoprostane were higher in asthmatic than in healthy children (18.3 pg/mL vs. 6.59 pg/mL, P < .026). They were also increased in children with moderate persistent asthma compared to those with episodic asthma (36.25 pg/mL and 12.28 pg/mL, P < .012), and in children with moderate persistent asthma and episodic asthma compared to healthy controls (36.25 pg/mL vs. 6.59 pg/mL [P < .0001] and 12.28 pg/mL versus 6.59 pg/mL [P < .0001], respectively). CONCLUSION: LTB4 and 8-isoprostane concentrations were increased in asthmatic children compared to healthy individuals, with differences detected for 2 degrees of asthma severity. Our findings suggest that EBC is a noninvasive method for airway inflammation and oxidative stress assessment.

Leptin on peritoneal macrophages of patients with endometriosis.

PROBLEM: The expression of cyclooxygenase (COX)-2 is considered as a marker of macrophage activation and has been implicated in the development of endometriosis. Leptin is an immunomodulator, which may also affect the development of endometriosis. However, how leptin contributes to these pathological processes has not been completely understood. The aim of this study was to investigate the effects of leptin on peritoneal macrophages and its relationship with endometriosis. METHODS OF STUDY: Peritoneal fluid from 60 women of reproductive age was obtained while they underwent laparoscopy. Forty patients had endometriosis and 20 patients did not have endometriosis. The concentration of leptin in the peritoneal fluid and prostaglandin F(2alpha) levels was measured by ELISA, and the other protein expression using Western blot when peritoneal macrophages were stimulated with leptin. RESULTS: Concentration of leptin in peritoneal fluid was increased in patients with endometriosis compared with disease-free normal control. Functional leptin receptor was present in peritoneal macrophages. Treatment of peritoneal macrophages with leptin induced COX-2 expression. Production of prostaglandin F(2alpha) by peritoneal macrophages was increased after leptin stimulation in women with endometriosis. CONCLUSION: Elevated concentration of leptin in peritoneal fluid may contribute to the pathological process of endometriosis through activation of peritoneal macrophages.

Potentially pathogenic bacteria cultured from the sputum of stable asthmatics are associated with increased 8-isoprostane and airway neutrophilia.

Potential bacterial pathogens are found in the airways in several diseases that are associated with neutrophilic inflammation. The aim of this study was to characterize subjects with stable asthma, with no symptoms of respiratory infection, to assess whether key potentially pathogenic bacteria were present in significant quantities in the airways and to correlate this with the pattern of airway inflammation and oxidative stress. Subjects with stable asthma (n = 115) and healthy controls (n = 8) underwent clinical assessment, including hypertonic saline challenge combined with sputum induction. A significant load of potentially pathogenic bacteria (> 10(6) cfu/mL) was cultured from the sputum of 17 (15%) subjects with stable asthma and was associated with higher total cell counts, proportion and number of neutrophils, sputum IL-8 and 8-isoprostane concentrations. The role of bacteria in potentiating neutrophilic asthma warrants further investigation. Therapies such as antibiotic and antioxidant treatment may be most effective in this sub-group of patients.

CREB mediates prostaglandin F2alpha-induced MUC5AC overexpression.

Mucus secretion is an important protective mechanism for the luminal lining of open tubular organs, but mucin overproduction in the respiratory tract can exacerbate the inflammatory process and cause airway obstruction. Production of MUC5AC, a predominant gel-forming mucin secreted by airway epithelia, can be induced by various inflammatory mediators such as prostaglandins. The two major prostaglandins involved in inflammation are PGE(2) and PGF(2alpha). PGE(2)-induced mucin production has been well studied, but the effect of PGF(2alpha) on mucin production remains poorly understood. To elucidate the effect and underlying mechanism of PGF(2alpha) on MUC5AC production, we investigated the signal transduction of PGF(2alpha) associated with this effect using normal human tracheobronchial epithelial cells. Our results demonstrated that PGF(2alpha) induces MUC5AC overproduction via a signaling cascade involving protein kinase C, ERK, p90 ribosomal S6 protein kinase, and CREB. The regulation of PGF(2alpha)-induced MUC5AC expression by CREB was further confirmed by cAMP response element-dependent MUC5AC promoter activity and by interaction between CREB and MUC5AC promoter. The abrogation of all downstream signaling activities via suppression of each signaling molecule along the pathway indicates that a single pathway from PGF(2alpha) receptor to CREB is responsible for inducing MUC5AC overproduction. As CREB also mediates mucin overproduction induced by PGE(2) and other inflammatory mediators, our findings have important clinical implications for the management of airway mucus hypersecretion.

Highly sensitive second antibody format enzymeimmunoassay for determination of 13,14-dihydro-15-keto-PGF2alpha in blood plasma of mithun (Bos frontalis).

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM) in blood plasma of mithun (Bos frontalis; bovine) on microtitreplates using second antibody coating technique and PGFM-horseradish peroxidase as a label has been developed. The wells of the microtitreplate were coated with affinity-purified goat IgG (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20microl plasma. The PGFM standard curve, with doses ranging from 0.1 to 50pg/well was linear. The sensitivity of the assay was 5pg/ml. PGFM standard curve in buffer showed parallelism with serially diluted mithun plasma containing high endogenous PGFM. Plasma PGFM concentrations estimated by using the developed EIA and commercially available PGFM EIA kit in the same samples were significantly correlated (r=0.98) and showed linearity. Intra- and inter-assay coefficients of variation were below 7%. Recovery of known concentrations of added PGFM in charcoal stripped plasma was linear (r=0.99). The developed EIA was further validated biologically by estimating PGFM in cyclic cows for the entire estrous cycle and in peri-parturient cows beginning day 7 prior to calving till day 30 post-calving; the concentrations were along with the expected lines as reported in bovine. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of PGFM directly in bovine plasma.

Constitutive and activation-inducible cyclooxygenase-2 expression enhances survival of chronic lymphocytic leukemia B cells.

We recently reported that activated normal human B lymphocytes express Cox-2. These findings prompted us to evaluate whether human B-CLL cells express Cox-2 and synthesize prostaglandins. In contrast to naive human B cells, B-CLL cells constitutively expressed Cox-2 protein and produced PGE2, PGF2alpha, and TXA2. Elevated Cox-2 expression was seen in a subgroup of B-CLL cells that exhibit poor prognostic factors, including unmutated variable heavy chain status and increased CD38 expression. Furthermore, stimulation of B-CLL cells with CD40 ligand plus TNFalpha increased Cox-2 levels. The role of Cox-2 in promoting B-CLL survival was investigated using nonselective and selective Cox-2 inhibitors. Significant reductions in B-CLL survival occurred following Cox-2 inhibition. These new findings support that constitutive Cox-2 expression in B-CLL cells promotes their survival and possibly their expansion in vivo. It will therefore be important to evaluate drugs that inhibit Cox-2 as potential therapeutic agents in B-CLL in vivo.

The oxygen stress index and levels of circulating interleukin-10 and interleukin-6 in patients with chronic heart failure.

OBJECTIVES: We sought to study circulating levels of pro- and anti-inflammatory cytokines together with the oxygen stress index in patients with chronic heart failure (CHF). BACKGROUND: Patients with CHF exhibit elevated levels of inflammatory and anti-inflammatory cytokines but the relative level of these cytokines with the oxygen stress index have not been reported. METHODS: Twenty-two patients with CHF and 10 control subjects were studied. Plasma levels of IL-6 and IL-10 were determined and the oxygen stress index was evaluated by urine 8-iso-PGF2alpha estimations. RESULTS: Plasma levels of IL-6 and IL-10 in CHF patients were significantly higher than those observed in the control subjects. Patients with more advanced disease (higher NYHA class) showed higher concentrations of IL-10 and IL-6 than those with less serious disease. 8-iso-PGF2alpha urine concentration (and therefore the oxygen stress index) was significantly higher in patients with CHF in comparison with control subjects. IL-6 plasma levels, but not IL-10 concentrations, correlated significantly with 8-iso-PGF2alpha levels in urine. CONCLUSIONS: Inflammatory and anti-inflammatory cytokine levels, as well as the oxidative stress index, are increased in patients with chronic heart failure. Inflammatory cytokine IL-6, but not anti-inflammatory cytokine Il-10, levels correlated significantly with the oxygen stress index.

Prostaglandin F2alpha- and FAS-activating antibody-induced regression of the corpus luteum involves caspase-8 and is defective in caspase-3 deficient mice.

We recently demonstrated that caspase-3 is important for apoptosis during spontaneous involution of the corpus luteum (CL). These studies tested if prostaglandin F2alpha (PGF2alpha) or FAS regulated luteal regression, utilize a caspase-3 dependent pathway to execute luteal cell apoptosis, and if the two receptors work via independent or potentially shared intracellular signaling components/pathways to activate caspase-3. Wild-type (WT) or caspase-3 deficient female mice, 25-26 days old, were given 10 IU equine chorionic gonadotropin (eCG) intraperitoneally (IP) followed by 10 IU human chorionic gonadotropin (hCG) IP 46 h later to synchronize ovulation. The animals were then injected with IgG (2 micrograms, i.v.), the FAS-activating antibody Jo2 (2 micrograms, i.v.), or PGF2alpha (10 micrograms, i.p.) at 24 or 48 h post-ovulation. Ovaries from each group were collected 8 h later for assessment of active caspase-3 enzyme and apoptosis (measured by the TUNEL assay) in the CL. Regardless of genotype or treatment, CL in ovaries collected from mice injected 24 h after ovulation showed no evidence of active caspase-3 or apoptosis. However, PGF2alpha or Jo2 at 48 h post-ovulation and collected 8 h later induced caspase-3 activation in 13.2 +/- 1.8% and 13.7 +/- 2.2 % of the cells, respectively and resulted in 16.35 +/- 0.7% (PGF2alpha) and 14.3 PlusMinus; 2.5% TUNEL-positive cells when compared to 1.48 +/- 0.8% of cells CL in IgG treated controls. In contrast, CL in ovaries collected from caspase-3 deficient mice whether treated with PGF2alpha, Jo2, or control IgG at 48 h post-ovulation showed little evidence of active caspase-3 or apoptosis. CL of WT mice treated with Jo2 at 48 h post-ovulation had an 8-fold increase in the activity of caspase-8, an activator of caspase-3 that is coupled to the FAS death receptor. Somewhat unexpectedly, however, treatment of WT mice with PGF2alpha at 48 h post-ovulation resulted in a 22-fold increase in caspase-8 activity in the CL, despite the fact that the receptor for PGF2alpha has not been shown to be directly coupled to caspase-8 recruitment and activation. We hypothesize that PGF2alpha initiates luteolysis in vivo, at least in part, by increasing the bioactivity or bioavailability of cytokines, such as FasL and that multiple endocrine factors work in concert to activate caspase-3-driven apoptosis during luteolysis.

[Immunoenzyme analysis of natural antibodies to endogenous bioregulators in patients with systemic lupus erythematosus]. / Immunofermentnyi analiz estestvennykh antitel k éndogennym bioreguliatoram u bol'nykh sistemnoi krasnoi volchankoi.

Serum levels of autoantibodies to endogenous bioregulators (prostaglandin F2 alpha, angiotensin II, epinephrine, bovine serum albumin, dinitrophenol) were measured in patients with systemic and integumental lupus erythematosus and donors and the diagnostic significance of deviations of these levels from the norm was evaluated. A total of 75 patients with lupus erythematosus aged 19-54 years with disease lasting for 0.5 to 18 months were examined. Significant differences between patients and donors were observed as regards virtually all parameters except IgG to angiotensin II.

Consumption of flavonoids in onions and black tea: lack of effect on F2-isoprostanes and autoantibodies to oxidized LDL in healthy humans.

BACKGROUND: Oxidative damage to lipids in vivo may be involved in the development of atherosclerosis and cancer. Onions and black tea are foods rich in flavonoids, predominantly the flavonoid quercetin, which is a potent in vitro inhibitor of membrane lipid peroxidation and LDL oxidation. OBJECTIVE: Our objective was to investigate the effects of consuming a high-flavonoid (HF) diet enriched with onions and black tea on indexes of oxidative damage in vivo compared with a low-flavonoid (LF) diet. DESIGN: Thirty-two healthy humans were studied in a randomized crossover design. Indexes of oxidative damage used were plasma F2-isoprostanes (a biomarker of lipid peroxidation in vivo) and the titer of antibodies to malondialdehyde (MDA)-modified LDL. RESULTS: There were no significant differences in the intake of macronutrients or assessed micronutrients, plasma F2-isoprostane concentrations, and MDA-LDL autoantibody titer between the HF and LF dietary treatments. In the men, however, plasma concentrations of the F2-isoprostane 8-epi-prostaglandin F2alpha were slightly higher after the HF treatment phase than after the LF treatment [0.31 +/- 0.029 nmol/L (111 +/- 10.4 ng/L) compared with 0.26 +/- 0.022 nmol/L (92 +/- 7.8 ng/L); P = 0.041]. In all subjects, plasma quercetin concentrations were significantly higher after the HF treatment phase than after the LF treatment: 221.6 +/- 37.4 nmol/L compared with less than the limit of detection of 66.2 nmol/L. CONCLUSION: Flavonoid consumption in onions and tea had no significant effect on plasma F2-isoprostane concentrations and MDA-LDL autoantibody titer in this study and thus does not seem to inhibit lipid peroxidation in humans.

Determination of 9alpha, 11beta prostaglandin F2 in human urine. combination of solid-phase extraction and radioimmunoassay.

This paper describes a new iodine-125 radioimmunoassay of 9alpha ,11beta-PGF2, and its use for the determination of urinary 9alpha,11beta-prostaglandin F2 after a selective one-step solid-phase extraction. The newly reported immunoassay is based on the use of 125I-tyrosyl methyl ester derivative of 9alpha,11beta-PGF2 and specific polyclonal antibody raised in rabbits. The assay detected as lowas 0.85 pg/tube 9alpha,11beta-PGF2, and the antibodyshowed lessthan 0.01 cross-reaction with PGF-ring metabolites (e.g., 8-iso-PGF2alpha, PGF2alpha 2,3-dinor-6-keto-PGF1alpha, and 5 more PGF-ring compounds). Both the intra-assay, and inter-assay CVs were lessthan 20% for internal controls containing low, medium and high concentrations of 9alpha,11beta-PGF2. Immuno-HPLC analysis showed a very low ratio of specific immunoreactivity in both non-extracted urine (6.5%), and in urine extracted on C18-silicacartridge (14.8%). By contrast, approximately 80% specific immunoreactivity could be achieved by using C2-silicaas the sorbent, acetonitrile: water (15:85, v/v) as wash solvent, and ethyl acetate as eluent of 9alpha,11beta-PGF2. This extraction procedure enabled a reasonably high extraction efficiency of 80.4 +/- 0.855 (mean +/- SEM, n=82), as determined by 3H-9alpha,11beta-PGF2. The new SPE/RIA method was applied for the determination of urinary 9alpha,11beta-PGF2 values in 50 healthy human volunteers. For the concentration and for the excretion rate 37.52 +/- 4.61 pg/ml (mean +/- SEM), and 3.50 + 0.35 ng/mmol creatinine (mean +/- SEM), respectively, was measured. The specificity of the SPE/RIA method was supported by the observed 69% decrease in 9alpha, 11beta-PGF2 excretion rate after acetylsalicylic acid treatment. The effect of nicotinic acid, a PGD2-stimulatory agent, was monitored by the urinary excretion of 9alpha ,11beta-PGF2 in 6 patients, by using the new SPE/RIA method. In patients responding with flushing symptoms nicotinic acid induced an increase of the urinary excretion of 9alpha,11beta-PGF2 in the range between 11% and 187%. In summary, the combination of the newly developed specific [125I] radioimmunoassay with solid-phase extraction on C2-silica cartridges enables the specific, sensitive, and reliable determination of 9alpha,11beta-PGF2 in human urine without the need for further laborious chromatographic purification before radioimmunoassay.

LPS mediated production of IL-1, PGE2 and PGF2alpha from term decidua involves tumour necrosis factor and tumour necrosis factor receptor p55.

Prostaglandins, with cytokines involved as intermediate factors, may have an essential role in premature labour when infection is present. We therefore wanted to study tumour necrosis factor (TNF), in cytokine and prostaglandin production in reproductive tissue. Decidual cell cultures were established and cells were stimulated with lipopolysaccharides (LPS). Media concentrations of TNF, interleukin-1 (IL-1), IL-6 and prostaglandin E2 and F2alpha were analysed, and involvement of LPS receptor CD14, TNF and TNF receptors (p55 and p75) were analysed, by studying effects after administration of specific antibodies. LPS induced an early peak elevation of TNF, with a subsequent release of IL-1, IL-6 and prostaglandins. Antibodies against CD14 inhibited these LPS effects. TNF antibodies reduced production of IL-1 and prostaglandins, whereas no significant influence on IL-6 production was observed. Antibodies against the TNF receptor p55 reduced all observed TNF effects. In contrast, p75 antibodies did not influence cytokine or prostaglandin production in this system. Our results suggest that increased TNF production is a prerequisite for LPS stimulated production of IL-1 and prostaglandins from decidual cells. LPS may directly stimulate IL-6 production. Of the two TNF receptors studied, only p55 seemed to be involved in the TNF signal transduction.

Radioimmunoassay of 15-keto-13,14-dihydro-prostaglandin F2alpha: an index for inflammation via cyclooxygenase catalysed lipid peroxidation.

The metabolism of PGF2alpha in human and other species results initially in the formation of 15-keto-dihydro-PGF2alpha and later to several beta-oxidized metabolites, which are species-specific. Since the discovery of cyclooxygenase-2 (COX-2), the importance of measuring various arachidonic acid metabolites during inflammatory conditions is on focus. This study presents the development and validation of a new radioimmunoassay of 15-keto-dihydro-PGF2alpha as an index of lipid peroxidation via cyclooxygenase (COX-1 and COX-2) pathway. Furthermore, its application in endotoxin-induced acute inflammation in pigs is presented. An antibody was raised in rabbits by immunization with 15-keto-dihydro-PGF2alpha coupled to BSA at the carboxylic acid by 1,1'-carbonyldiimidazole method. The cross-reactivity of the antibody with PGF2alpha, 15-keto-PGF2alpha, PGE2, 15-keto-13,14-dihydro-PGE2, 8-iso-15-keto-13,14-dihydro-PGF2alpha, 11beta-PGF2alpha, 9beta-PGF2alpha, TXB2 and 8-iso-PGF3alpha was 0.02, 0.43, < 0.001, 0.5, 1.7, < 0.001, < 0.001, < 0.001, 0.01%, respectively. The intra-assay precision was 12.2% (CV) at the level of 64 pg/0.1 ml and 14.0% with 512 pg/0.1 ml in the human plasma. Similarly, intra-assay accuracy was 108.6% and 103.3% for the low and the high standards, respectively. The detection limit was about 45 pmol/L. 15-keto-dihydro-PGF2alpha levels in plasma from normal human volunteers were evaluated and found to correlate with the obtained values by GC-MS methods from other studies. The levels of 15-keto-dihydro-PGF2alpha in the plasma increased several-fold after endotoxin infusion (10 microg/kg/h over 6 h) to the pigs. Thus, this 1 5-keto-dihydro-PGF2alpha radioimmunoassay method is relevant to apply in inflammatory injury, and other physiological and pathophysiological studies, as an index of in vivo enzymatic lipid peroxidation.

Radioimmunoassay of 8-iso-prostaglandin F2alpha: an index for oxidative injury via free radical catalysed lipid peroxidation.

8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), a major F2-isoprostane, is biosynthesized in vivo through nonenzymatic free radical-catalysed peroxidation of arachidonic acid. The levels of F2-isoprostanes both free in the circulation and esterified to the tissue phospholipids increase intensely in animal models of oxidant injury. This study presents the development and validation of a radioimmunoassay of 8-iso-PGF2alpha for the measurement of this substance in the body fluids. Furthermore, its application in normal human volunteers, a pharmacokinetic study performed in rabbits with 8-iso-PGF2alpha, and hepatic lipid peroxidation in rats is reported. An antibody was raised in rabbits by immunization with 8-iso-PGF2alpha coupled to BSA at the carboxylic acid by 1,1'-Carbonyldiimmidazole method. The cross-reactivity of the antibody with 8-iso-15-keto-13,14-dihydro-PGF2alpha, 8-iso-PGF2beta, PGF2alpha, 15-keto-PGF2alpha, 15-keto-13,14-dihydro-PGF2alpha,TXB2, 11beta-PGF2alpha, 9beta-PGF2alpha and 8-iso-PGF3alpha was 1.7, 9.8, 1.1, 0.01, 0.01, 0.1, 0.03, 1.8 and 0.6%, respectively. The intraassay precision was 14.5% (CV) at the level of 64 pg/0.1 ml and 12.2% with 512 pg/0.1 ml in the human plasma. Similarly, intra-assay accuracy was 95.6% and 101% for the low and the high standard, respectively. The detection limit was about 23 pmol/l. The appearance and disappearance of 8-iso-PGF2alpha in the blood and urine following intravenous administration of 8-iso-PGF2alpha in the rabbit was rapid. Free 8-iso-PGF2alpha levels in plasma and urine from normal human volunteers are evaluated and found to correlate with the obtained values by gas chromatography-mass spectrometry methods from other studies. The levels of free 8-iso-PGF2alpha in the plasma and urine increased 7- and 102-fold, respectively, after CCl4 administration to rats. Thus, this 8-iso PGF2alpha radioimmunoassay method is relevant to apply in the oxidative injury studies as an index of in vivo lipid peroxidation through free radical catalysis mechanism.

[Effect of diet therapy on metabolism of prostaglandins in patients with hypertension and obesity]. / Vliianie dietoterapii na obmen prostaglandinov yu bol'nykh gipertonicheskoi bolezn'iu i ozhireniem.

Significant disturbances of immune regulation of prostaglandine metabolism were found in patients with hypertension and obesity. Use of antisclerotic diet with low sodium contents promoted positive changes of clinical symptoms of diseases, in particular normalizations of process of immune regulation of prostaglandine metabolism connected with formation of natural antibodies.

Histamine and prostaglandin interaction in the central regulation of ACTH secretion.

Since prostaglandins (PGs) and histamine (HA) seem to be involved in the activation of the hypothalamic-pituitary-adrenal axis following immunochallenges with lipopolysaccharide (LPS) endotoxin and cytokines, we investigated whether the histaminergic and eicosanoid systems in the male rat brain interact in their regulation of ACTH secretion. The PG synthesis inhibitor indomethacin (10 mg/kg i.p.) attenuated the ACTH response to LPS (10 micrograms/kg i.p.) and HA (30 micrograms i.c.v.). Infusion of PGE1, PGE2 or PGF2 alpha (1 and/or 5 micrograms infused intracerebroventricularly) stimulated ACTH secretion. The ACTH response to PGE1 was inhibited by the HA synthesis inhibitor alpha-fluoromethyl-histidine and the H1 receptor antagonist mepyramine (MEP) but not the H2 receptor antagonist cimetidine (CIM). Neither MEP nor CIM affected the ACTH response to PGE2. MEP and CIM attenuated the ACTH response induced by PGF2 alpha. The findings indicate that HA and some PGs in the brain interact in their stimulatory regulation of ACTH secretion. Such an interaction may also be involved in their mediation of the ACTH response to immunochallenges.

Induction of hydrometra in goats by means of active immunization against prostaglandin F2 alpha.

Hydrometra is a pathological condition of the uterus which is characterized by accumulation of aseptic fluid in the presence of a persistent corpus luteum. It forms a major cause of subfertility in goats. Active immunization against prostaglandin F2 alpha (PGF2 alpha), the luteolytic hormone in this species, was used to explore the possibility for artificial induction of this pseudopregnant condition. During the breeding season, 11 goats (group I) were immunized with 5 mg PGF2 alpha-ovalbumin conjugate in Freund's complete adjuvant, 4 goats were control-immunized (group CI) and 5 goats remained untreated (group C). Jugular blood samples were taken twice a week (monday and thursday) for measurements of plasma progesterone and binding of 3HPGF2 alpha. In conjunction with blood sampling, transcutaneous ultrasonographic examination of the uterus took place to detect the presence of fluid in the uterus. Before and immediately after immunization, the mean (+/-SD) duration of luteal phases (progesterone concentrations > or = 1 ng ml-1) was 16.7 +/- 1.6 (n = 39), 17.8 +/- 1.3 (n = 23) and 16.9 +/- 1.1 (n = 18) days in animals of group I, CI and C respectively. Ten goats of group I developed an antibody titre. Persistence of luteal function (mean duration +/-SD: 150.3 +/- 23.5 days) occurred in 6 of these animals and in 1 goat of group CI. Accumulation of fluid in the uterus in group I was first observed between day 31 and 38 of the prolonged luteal phase. Discharge of uterine fluid occurred as soon as the plasma progesterone concentration reached a level lower than 0.5 ng ml-1. It is concluded that immunization against PGF2 alpha is an effective method to induce pseudopregnancy in goats, providing a model for studies on luteal maintenance and uterine function in the absence of a conceptus.